Live attenuated, replicating vaccines, rather than inactivated preparations, have provided the most effective protection against viral infection and disease. These vaccines elicit essentially life-long protective immunity. In contrast, immunity induced by inactivated or subunit vaccines is generally of more limited duration. A key factor in pursuit of the latter approaches is safety. An overview of replicating and non-replicating viral vectors for vaccine development is given in the publication of Marjorie Robert-Guroff, Replicating and Non-replicating Viral Vectors for Vaccine Development, Curr. Opin. Biotechnol. 18:546-556, 2007.
Recombinant viruses are widely used to express foreign antigens in infected cells. Specifically, recombinant poxviruses are currently tested as promising vaccines to induce an immune response against a foreign antigen expressed from the poxvirus vector. Most popular are avipoxviruses on the one side and vaccinia viruses (VACV) on the other side. U.S. Pat. No. 5,736,368 and U.S. Pat. No. 6,051,410 disclose recombinant vaccinia virus strain Wyeth which expresses HIV antigens and proteins. U.S. Pat. No. 5,747,324 discloses a recombinant VACV strain NYCBH expressing lentivirus genes. EP 0 243 029 discloses a recombinant VACV strain Western Reserve expressing human retrovirus genes. For the expression of heterologous genes in poxviruses several promoters are known to the person skilled in the art, such as the 30K and 40K promoters (see, e.g., U.S. Pat. No. 5,747,324), a strong synthetic early/late promoter (see, e.g., Sutter et al., A recombinant vector derived from the host range-restricted and highly attenuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus, Vaccine 12,1032-40, 1994), the p7.5 promoter (see, e.g., Endo et al., Homotypic and heterotypic protection against influenza virus infection in mice by recombinant vaccinia virus expressing the haemagglutinin or nucleoprotein of influenza virus, J. Gen. Virol. 72, 699-703, 1991) and the promoter derived from the cowpox virus A-type inclusion (ATI) gene (Li et al., High-level expression of Amsacta moorei entomopoxvirus spheroidin depends on sequences within the gene, J. Gen. Virol. 79,613, 1998). All of these promoters have been used in recombinant VACV to express heterologous genes and were shown to express said genes very efficiently resulting in relatively high amounts of the protein encoded by the heterologous gene. In general, for many vaccination approaches it is highly desired that the antigen against which an immune response is to be induced is expressed in high amounts.
Induction of a strong humoral and cellular immune response against a foreign gene product expressed by, e.g., a VACV vector is hampered by the fact that the foreign gene product has to compete with all of the more than 150 antigens of the VACV vector for recognition and induction of specific antibodies and T cells. Immunodominance of vector CD8 T cell epitopes prevents induction of a strong CD8 T cell response against the foreign gene product. (Smith et al., Immunodominance of poxyiral-specific CTL in a human trial of recombinant-modified vaccinia Ankara. J. Immunol. 175:8431-8437, 2005.) This applies to replicating VACV vectors such as Dryvax, as well as to replication deficient vectors like NYVAC and Modified Vaccinia virus Ankara, MVA.
For expression of a recombinant antigen by VACV poxvirus-specific promoters but not common eukaryotic promoters may be used. The reason for this is the specific biology of poxviruses which replicate in the cytoplasm and bring their own, cell-autonomous transcriptional machinery with them that does not recognize typical eukaryotic promoters.
The viral replication cycle is divided into two major phases, an early phase comprising the first two hours after infection before DNA replication, and a late phase starting at the onset of viral DNA replication at 2-4 hours after infection. The late phase spans the rest of the viral replication cycle from ˜2-20 h after infection until progeny virus is released from the infected cell. There are a number of poxyiral promoter types which are distinguished and named by the time periods within the viral replication cycle in which they are active, for example, early and late promoters. (See, e.g., Davison and Moss, Structure of Vaccinia Virus Late Promoters, J. Mol. Biol. 210:771-784, 1989; Davison and Moss, Structure of Vaccinia Virus Early Promoters, J. Mol. Biol. 210:749-769, 1989; and Hirschmann et al., Mutational Analysis of a Vaccinia Virus Intermediate Promoter in vitro and in vivo, Journal of Virology 64:6063-6069, 1990, all of which are hereby incorporated by reference.)
Whereas early promoters can also be active late in infection, activity of late promoters is confined to the late phase. A third class of promoters, named intermediate promoters, is active at the transition of early to late phase and is dependent on viral DNA replication. The latter also applies to late promoters, however, transcription from intermediate promoters starts earlier than from typical late promoters and requires a different set of transcription factors.
It became increasingly clear over recent years that the choice of the temporal class of poxyiral promoter for antigen expression has profound effects on the strength and quality of the antigen-specific immune response. It was shown that T cell responses against antigens expressed under the control of a late promoter are weaker than those obtained with the same antigen under the control of an early promoter. (Bronte et al., Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine. Proc. Natl. Acad. Sci. U.S.A 94:3183-3188, 1997; Coupar et al., Temporal regulation of influenza hemagglutinin expression in vaccinia virus recombinants and effects on the immune response. Eur. J. Immunol. 16:1479-1487, 1986.)
Even more strikingly, it was shown that in repeated autologous immunizations with VACV as well as with the replication-defective VACV vector MVA, recall CD8 T cell responses against antigens under the control of an exclusively late promoter can fail completely. This failure resulted in an almost undetectable antigen-specific CD8 T cell response after the second immunization (Kastenmuller et al., Cross-competition of CD8+ T cells shapes the immunodominance hierarchy during boost vaccination. J. Exp. Med. 204:2187-2198, 2007.)
Thus, early expression of antigens by VACV vectors appears to be crucial for efficient antigen-specific CD8 T cell responses. It has also been shown that an early-expressed VACV vector antigen not only competes with late expressed antigens but also with other early antigens for immunodominance in the CD8 T cell response (Kastenmuller et al., Cross-competition of CD8+ T cells shapes the immunodominance hierarchy during boost vaccination, J. Exp. Med. 204:2187-2198, 2007). The specific properties of the early portion of the poxyiral promoter might thus be important for induction of an antigen-specific T cell response. Moreover, it is a commonly held view and a general rule that higher amounts of antigen are beneficial for induction of stronger antigen-specific immune responses (for the poxvirus field, see for example Wyatt et al., Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine 26:486-493, 2008.)
A promoter combining 4 early promoter elements and a late promoter element from the ATI gene has been described previously and has been shown to direct increased early antigen expression (Funahashi et al., Increased expression in vivo and in vitro of foreign genes directed by A-type inclusion body hybrid promoters in recombinant vaccinia viruses. J. Virol. 65:5584-5588, 1991; Wyatt et al., Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines. Vaccine 26:486-493, 2008). T cell responses induced by an antigen driven by such a promoter in a recombinant replication competent vaccinia virus vector have been analyzed after a single immunization and were, however, found to be only slightly different from those obtained with the classical p7.5 promoter in this setting. (Funahashi et al., 1991.)
Jin et al. (Constructions of vaccinia virus A-type inclusion body protein, tandemly repeated mutant 7.5 kDa protein, and hemagglutinin gene promoters support high levels of expression, Arch. Virol. 138:315-330, 1994) reported the construction of recombinant VACV harbouring promoters consisting of a VACV ATI promoter combined with tandem repeats (2 to 38 copies) of a mutated p7.5 promoter operably linked to the CAT gene. Up to 10-15 repetitions of the mutated p7.5 promoter appeared to be effective in increasing early gene expression. However, with all constructs, the amount of CAT protein produced in the presence of cytosine arabinoside (AraC) (i.e. when the viral replication cycle was arrested in the early phase) was only less than one-tenth of the amount produced in the absence of AraC, indicating that although early gene expression was increased, most of the expressed antigen was obviously produced during the late phase of infection.
Accordingly, there is a need for improved viral vectors that enable early expression of foreign antigens and induction of a strong antigen-specific immune response.